Immunoregulatory T cells (Treg cells) have recently been identified as an active mechanism of immune suppression for maintaining T cell tolerance. Among several sets of Treg cells described, CD4+CD25+ Treg cells comprise 5-10% of peripheral CD4+ T cells. These cells, identified in mice and humans, exhibit potent immunoregulatory functions and are considered "professional regulatory T cells". In mouse models, transfer of CD4+CD25+ Treg cells can suppress autoimmune diseases. Treg cells have also been implicated in the regulation of anti-tumor immunity, graft-versus-host disease (GVHD) associated with transplantation, and anti-microbial immunity. Thus, the survival or death of CD4+CD25+ Treg cells is likely to be important for immune homeostasis. The mechanisms that govern CD4+CD25+ Treg cell survival remains elusive, although the generation of CD4+CD25+ Treg cells is CD28-dependent. In preliminary studies, several Bcl-x isoforms were identified including a novel Bcl-x isoform, Bcl-x-gamma, which is characterized by a unique C-terminus and inhibits T cell apoptosis after TCR ligation. These results, as well as the reports of others, further showed that CD28 co-stimulatory signals can up-regulate the expression of the anti-apoptotic proteins Bcl-xL and Bcl-x-gamma, suggesting that Bcl-xgamma and Bcl-xL promote T cell survival after CD3/CD28 co-ligation. This proposal will test the hypothesis that CD4+CD25+ Treg cells have a specific survival program in which Bcl-x genes play an important role through CD28. The corollary hypothesis is that a shift toward survival, rather than apoptosis, in CD4+CD25+ Treg cells may lead to reduced autoimmunity, decreased GVHD, and diminished, deleterious tissue damage associated with anti-microbial immunity. The primary goal of this proposal is to define the survival program of CD4+CD25+ Treg cells, and especially the roles of Bcl-x gene family members. This goal will be pursued through the following specific aims: (1) To determine the function of Bcl-x proteins in CD4+CD25+ Treg cells by analysis of the expression and pro-survival function of these genes in the Treg cells; (2) To determine whether Bcl-x up-regulation is responsible for the anergic state of CD4+CD25+ Treg cells; and (3) To determine the potential for CD4+CD25+ Treg cells, rescued from death by transfection with Bcl-x-gamma and Bcl-xL, to reduce the immunopathology in a mouse model of autoimmune gastritis. Molecular definition of the survival program of CD4+CD25+ Treg cells may lead to development of novel therapeutic strategies for autoimmune diseases, as well as viral and bacterial infections by specifically promoting Treg cell survival.